Asymmetrical flow field-flow fractionation to probe the dynamic association equilibria of β-D-galactosidase

Iro K. Ventouri*, Alina Astefanei, Erwin R. Kaal, Rob Haselberg, Govert W. Somsen, Peter J. Schoenmakers

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Protein dynamics play a significant role in many aspects of enzyme activity. Monitoring of structural changes and aggregation of biotechnological enzymes under native conditions is important to safeguard their properties and function. In this work, the potential of asymmetrical flow field-flow fractionation (AF4) to study the dynamic association equilibria of the enzyme β-D-galactosidase (β-D-Gal) was evaluated. Three commercial products of β-D-Gal were investigated using carrier liquids containing sodium chloride or ammonium acetate, and the effect of adding magnesium (II) chloride to the carrier liquid was assessed. Preservation of protein structural integrity during AF4 analysis was essential and the influence of several parameters, such as the focusing step (including use of frit-inlet), cross flow, and injected amount, was studied. Size-exclusion chromatography (SEC) and dynamic light scattering (DLS) were used to corroborate the in-solution enzyme oligomerization observed with AF4. In contrast to SEC, AF4 provided sufficiently mild separation conditions to monitor protein conformations without disturbing the dynamic association equilibria. AF4 analysis showed that ammonium acetate concentrations above 40 mM led to further association of the dimers (“tetramerization”) of β-D-Gal. Magnesium ions, which are needed to activate β-D-Gal, appeared to induce dimer association, raising justifiable questions about the role of divalent metal ions in protein oligomerization and on whether tetramers or dimers are the most active form of β-D-Gal.

Original languageEnglish
Article number461719
Pages (from-to)10
Number of pages10
JournalJournal of Chromatography A
Early online date13 Nov 2020
Publication statusPublished - 4 Jan 2021


Iro K. Ventouri acknowledges the HOSAna project, which is funded by the Netherlands Organization for Scientific Research (NWO) in the framework of the Programmatic Technology Area PTA-COAST4 of the Fund New Chemical Innovations (project nr. 053.21.117).

FundersFunder number
DSM Biotechnology Center
Fund New Chemical Innovations053.21.117
Postnova Analytics
Nederlandse Organisatie voor Wetenschappelijk Onderzoek


    • enzyme β-D-galactosidase
    • Field-Flow Fractionation
    • frit-inlet AF4
    • protein association equilibria


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